Immunohistochemical Analysis of Pygo2 and E-cadherin in HCC

Tissues were fixed with 10% neutral formalin, embedded in paraffin, and 3-μm-thick sections were prepared by pathological technologist. Hematoxylin-eosin (HE) stain was performed as previous describe [17 ]. For immunohistochemistry (IHC) staining, sections were deparaffinized, hydrated and soaked in 3% H₂O₂ for 15 minutes at room temperature, and then incubated with Pygo2 polyclonal antibody (1:4000, ab109001, Abcam) and E-cadherin (1:1000, 20874-1-AP, Proteintech) at 4°C overnight. Biotinylated secondary antibody and diaminobenzidine were purchased from Maixin Biotechnology (Fuzhou, China). Evaluation of Pygo2 and E-cadherin staining in HCC tissue sections was performed based on the IHC assessment methods used by Popadiuk et al. for epithelial ovarian cancer [12 (link)] and Motoyuki Hashiguchi et al. for HCC [44 (link)], respectively.